Life on Minerals: Binding Behaviors of Oligonucleotides on Zirconium Silicate and Its Inhibitory Activity for the Self-Cleavage of Hammerhead Ribozyme

Abstract

The role of minerals in the chemical evolution of RNA molecules is an important issue when considering the early stage of the Hadean Earth. In particular, the interaction between functional ribozymes and ancient minerals under simulated primitive conditions is a recent research focus. We are currently attempting to design a primitive RNA metabolic network which would function with minerals, and believe that the simulated chemical network of RNA molecules would be useful for evaluation of the chemical evolution from a simple RNA mixture to an RNA-based life-like system. First, we measured the binding interactions of oligonucleotides with four types of minerals; Aerosil silica, zirconium silicate, sepiolite, and montmorillonite. Oligonucleotides bound zirconium silicate and montmorillonite in the presence of MgCl₂, and bound sepiolite both in the presence and absence of MgCl₂, but they did not bind Aerosil. Based on the binding behavior, we attempted the self-cleavage reaction of the hammerhead ribozyme from an avocado viroid. This reaction was strongly inhibited by zirconium silicate, a compound regarded as mineral evidence for the existence of water. The present study suggests that the chemical evolution of functional RNA molecules requires specific conformational binding, resulting in efficient ribozyme function as well as zirconium silicate for the chemical evolution of biomolecules.

Keywords: Aerosil; Hadean Earth; adsorption; chemical evolution; hammerhead ribozyme; mineral; montmorillonite; sepiolite; zirconium silicate.

Figure 1

Schematic model for a primitive RNA metabolic system on a mineral surface. Short RNA molecules do not bind onto minerals strongly, while functional long RNA molecules bind onto minerals strongly. The functional RNA molecules could form a metabolic chemical network on the mineral. This can be regarded as an integrated RNA system prior to a cell-type compartmentation of RNA molecules.

Figure 2

Secondary structures estimated using RNAfold WebServer for some oligonucleotides. (a) RNA molecules: 1: ASBVd79(−):HHR, 2: rna-ASBVd45, 3: rna-ASBVd45-^3′(GA)₅, 4: rna-ASBVd45-^3′C₁₀, 5: rna-ASBVd45-^5′(GA)₅, 6: rna-ASBVd45-^5′C₁₀; ((b) right) DNA molecules: 1: dna-ASBVd45, 2: dna-ASBVd45-^5′(GA)₅, 3: dna-ASBVd45-^5′C₁₀, 4: dna-ASBVd45-^3′C₁₀, 5: dna-ASBVd45-^5′A₁₀, 6: dna-ASBVd45-^3′(GA)₅, 7: dna-ASBVd45-^3′(GA)₁₀. The calculations were carried out at 328.15 K for RNA and 298.15 K for DNA. Most parameters were applied as default values, such as maximum loop size at 30, maximum energy difference at 10%, maximum number of structures at 20, and minimum helix length at 3. The predicted lowest free energy structures are displayed on the basis of the lowest free energy structure since the structures are basically the same as the structure of composed of highly probable base pairs.

Figure 3

Adsorbed amounts of dna-ASBVd45 on minerals in the presence and absence of MgCl₂. Mineral phase/aqueous phase: 20.0 mg/2000 µL, [dna-ASBVd45] = 1.14 µM, [HEPES] = 0.05 M, pH: 7.5, [MgCl₂]: (a) 0.05 M, (b) 0 M. Adsorption time: 1 h (blue bars), 24 h (red bars).

Figure 4

Adsorbed fractions of different model oligonucleotides (DNA) on zirconium silicate in the presence of MgCl₂. Mineral phase/aqueous phase: 10.0 mg/2000 μL, Solutions: [DNA] = 1.0 μM, [HEPES] = 0.05 M, pH: 7.5, [MgCl₂] = 0.05 M. Adsorption time: 1 h (blue bars), 24 h (red bars).

Figure 5

Adsorbed fraction of oligo(A) on minerals in the presence and absence of MgCl₂. Mineral phase/aqueous phase: 20.0 mg/2000 µL, [oligoA] = 48 µM, [HEPES] = 0.05 M, pH: 7.5, [MgCl₂]: (a) 0.05 M; (b) 0 M. Adsorption time: 1 h (blue bars), 24 h (red bars).

Figure 6

Adsorption isotherm for binding of oligo(A) on zirconium silicate. Mineral phase/aqueous phase: 10.0 mg/2000 µL, [oligoA] = 2.86–116 µM, [HEPES] = 0.05 M, pH: 7.5, [MgCl₂] = 0.05 M, 24 h.

Figure 7

Adsorbed fractions of different model oligonucleotides (RNA) on zirconium silicate in the presence of MgCl₂. Mineral phase/aqueous phase: 20.0 mg/2000 µL, [RNA molecules] = 20 µg/2000 µL, [HEPES] = 0.05 M, pH: 7.5, [MgCl₂] = 0.05 M. After centrifugation, the precipitate was washed for 1 h with 1600 µL of 0.0625 M EDTA solution.

Figure 8

Self-cleavage degree in the presence of zirconium silicate (open circles) and montmorillonite (gray circles). Reaction conditions: Mineral phase/aqueous phase: 0–10 mg/50 µL solution, [ASBVd(−):HHR] = 3 µg/50 µL, [HEPES] = 0.05 M, pH: 7.5, [MgCl₂] = 0.05 M, 55 °C, reaction time: 120 min. The reaction was stopped and washed by addition of 170 µL of 0.029 M EDTA solution (pH = 8.0) twice. The products were analyzed by HPLC, and the self-cleavage degree was calculated by combining the washed solutions.

Figure 9

Reaction kinetic profile for the self-cleavage of ASBVd(−):HHR in the presence of zirconium silicate and montmorillonite. Reaction conditions without mineral: [ASBVd(−):HHR] = 3 µg/50 µL, [HEPES] = 0.05 M, pH: 7.5, [MgCl₂] = 0.05 M, 55 °C, reaction time: 0–90 min. Reaction conditions with minerals: Mineral phase/aqueous phase: 2.5 mg/50 µL solution, [ASBVd(−):HHR] = 3 µg/50 µL, [HEPES] = 0.05 M, pH: 7.5, [MgCl₂] = 0.05 M, 55 °C, reaction time: 0–90 min. The reaction was stopped and washed by addition of 170 µL of 0.029 M EDTA solution (pH = 8.0) two times. The washing solutions were analyzed by HPLC, and the self-cleavage degree was calculated by combining the washed solutions. Black open circles: control, red open circles: zirconium silicate, blue open circles: montmorillonite. Dashed lines indicate fitted lines based on the rate constants shown in Table 4.